A kind of purification method of lithocholic acid

Abstract

The present invention provides a method for refining lithocholic acid, which comprises the steps of: reacting crude lithocholic acid with a base in water, then adding dichloromethane to mix, adding an alcohol solvent to the aqueous phase after layering, then adding an acid to adjust the pH to 3-4, precipitating a solid, and performing solid-liquid separation to obtain high-purity lithocholic acid; wherein the base is an inorganic potassium salt and / or an inorganic sodium salt. The method of the present invention can purify crude lithocholic acid to obtain high-purity lithocholic acid, and the method is simple and has a high product yield.

Description

Technical Field

[0001] The present invention relates to the technical field of drug purification, and more specifically, to a method for refining lithocholic acid. Background Art

[0002] Lithocholic acid (3α-hydroxy-5β-azane acid, compound 1 below) is a secondary bile acid, also known as cholelithic acid, which exists in the bile of higher vertebrates. The change of its content has important reference value in the diagnosis of liver diseases. Lithocholic acid has a wide range of biological activities, such as activating vitamin D receptors, inhibiting the activity of diabetes-related target PTP1B, activating TGR5 receptors, which are related targets for the treatment of diabetes, antibacterial and antifungal activities, and anti-aging activities. In particular, the compound also has anti-tumor activity. In recent years, studies have found that lithocholic acid can inhibit the mutation of DNA polymerase β and reduce the probability of tumors caused by DNA polymerase β mutations;

[0003]

[0004] Lithocholic acid (LCA) is a combined bile acid synthesized by bile acid and lithocholic acid in the liver. It is one of the main bile acid components. The main source is currently extracted from animal bile, but there are many impurities in the extraction process, making the extract a crude lithocholic acid with a purity of only about 90%, which mainly includes the following isomeric impurities such as compound 2 and compound 3;

[0005]

[0006] The existing purification method mainly utilizes organic solvents to purify lithocholic acid and impurities according to their different solubilities. However, due to the small difference between the solubility of lithocholic acid and the solubility of impurities, the purification method has a low yield and low purity, which cannot meet the purity requirements of the standard product. Summary of the invention

[0007] Based on this, in order to solve the problem of low purity of crude lithocholic acid extracted from animal bile in the prior art, the present invention provides a simple method for refining lithocholic acid. After purifying the crude lithocholic acid by this method, high-purity lithocholic acid with a purity of ≥99.1% can be obtained, and the yield is relatively high.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] A method for refining lithocholic acid comprises the following steps:

[0010] The crude lithocholic acid reacts with a base in water, then dichloromethane is added to mix, an alcohol solvent is added to the aqueous phase after separation, and then an acid is added to adjust the pH to 3-4, solids are precipitated, and solid-liquid separation is performed to obtain high-purity lithocholic acid;

[0011] Wherein, the base is an inorganic potassium salt and / or an inorganic sodium salt.

[0012] In some embodiments, the mass ratio of the crude lithocholic acid to the base is 1:1 to 2.5.

[0013] In some embodiments, the inorganic potassium salt is potassium hydroxide and / or potassium carbonate; the inorganic sodium salt is sodium hydroxide and / or sodium carbonate.

[0014] In some embodiments, the alcohol solvent is ethanol.

[0015] In some embodiments, the acid is hydrochloric acid.

[0016] In some embodiments, the temperature at which the crude lithocholic acid and the base react in water is 20-30°C.

[0017] In some embodiments, the purity of the crude lithocholic acid is 85-95%.

[0018] In some embodiments, the crude lithocholic acid comprises a first compound of the following formula I and / or a second compound of the following formula II:

[0019]

[0020] Specifically, the refining method comprises the following steps:

[0021] The crude lithocholic acid is added to water, and the base is added at 20-30°C, and the pH is adjusted to 9-10 for reaction until the reaction solution is clear, and then dichloromethane is added for washing at least twice. After stratification, an alcohol solvent is added to the aqueous phase at a temperature of 10-20°C, and then an acid is added to adjust the pH to 3-4, solids are precipitated, and solid-liquid separation is performed to obtain high-purity lithocholic acid.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] According to the invention, crude lithocholic acid is reacted with an alkali to generate a potassium salt or a sodium salt, wherein the solubility of the potassium salt or the sodium salt in water is significantly higher than that of lithocholic acid and its isomers, and impurities which cannot react with the alkali and are insoluble in water are effectively removed; then dichloromethane is added for washing, and the impurities whose solubility in dichloromethane is higher than that in water are further removed by dichloromethane extraction; after phase separation, an alcohol solvent is added to the aqueous phase and the pH value is adjusted to carboxylate the potassium salt or the sodium salt and convert it into lithocholic acid; at the same time, under non-heating conditions, the solubility of lithocholic acid and its isomers in the alcohol solvent is low, and lithocholic acid is precipitated due to its high concentration, and high-purity lithocholic acid is obtained after solid-liquid separation.

[0024] The purity of the lithocholic acid obtained by the method of the invention is ≥99.1%, and the yield can reach 84.5%. DETAILED DESCRIPTION

[0025] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] Example 1

[0028] A method for refining lithocholic acid, the reaction process of which is as follows:

[0029]

[0030] The specific steps include:

[0031] Add 50g of crude lithocholic acid (purity of 90%±2%) to a reaction flask, then add 300mL of water, add dropwise a 10% sodium hydroxide solution at 25°C, adjust the pH to 10 and react until the reaction solution is clear; then add dichloromethane to the reaction solution for washing twice, 100mL each time, add 90mL of ethanol to the aqueous phase after stratification, control the temperature at 20°C, add dropwise 1mol / L hydrochloric acid to adjust the pH to 3, wait until the precipitated solid no longer increases, separate the solid and liquid, wash the filter cake with a small amount of methanol, and dry the solid to obtain 41.2g of lithocholic acid.

[0032] The lithocholic acid obtained by the method of this example has a yield of 82.4% and a purity of 99.6%.

[0033] Example 2

[0034] A method for refining lithocholic acid comprises the following steps:

[0035] Add 20g of crude lithocholic acid (purity of 90%±2%) to the reaction flask, then add 120mL of water, add dropwise a 10% potassium hydroxide solution at 30°C, adjust the pH to 9 and react until the reaction solution is clear; then add dichloromethane to the reaction solution for washing twice, 40mL each time, add 35mL of ethanol to the aqueous phase after stratification, control the temperature at 15°C, add dropwise 1mol / L hydrochloric acid to adjust the pH to 4, wait until the precipitated solid no longer increases, separate the solid and liquid, wash the filter cake with a small amount of methanol, and dry the solid to obtain 15.5g of lithocholic acid.

[0036] The lithocholic acid obtained by the method of this example has a yield of 77.4% and a purity of 99.3%.

[0037] Example 3

[0038] A method for refining lithocholic acid comprises the following steps:

[0039] Add 40g of crude lithocholic acid (purity of 90%±2%) to a reaction flask, then add 240mL of water, add dropwise a 10% sodium hydroxide solution at 20°C, adjust the pH to 10 and react until the reaction solution is clear; then add dichloromethane to the reaction solution for washing twice, 80mL each time, add 70mL of ethanol to the aqueous phase after stratification, control the temperature at 10°C, add dropwise 1mol / L hydrochloric acid to adjust the pH to 3, wait until the precipitated solid no longer increases, separate the solid and liquid, wash the filter cake with a small amount of methanol, and dry the solid to obtain 33.8g of lithocholic acid.

[0040] The lithocholic acid obtained by the method of this example has a yield of 84.5% and a purity of 99.1%.

[0041] Comparative Example 1

[0042] A method for refining lithocholic acid comprises the following steps:

[0043] Add 50 g of crude lithocholic acid (purity: 90% ± 2%) into a reaction flask, then add 800 mL of ethyl acetate and 800 mL of dichloromethane to dissolve, concentrate and distill to remove dichloromethane, cool, filter, and dry the solid to obtain 38.6 g of lithocholic acid, with a yield of 79.2% and a purity of 97.1%.

[0044] Comparative Example 2

[0045] A method for refining lithocholic acid comprises the following steps:

[0046] Add 40g crude lithocholic acid (purity 90%±2%) to the reaction bottle, then add 240mL water, add 10% sodium hydroxide solution at 20°C, adjust pH to 10 and react until the reaction solution is clear; after separation, add 70mL ethanol to the aqueous phase, control the temperature at 10°C and add 1mol / L hydrochloric acid to adjust pH to 3, wait until the precipitated solid no longer increases, separate the solid and liquid, wash the filter cake with a small amount of methanol, and dry the solid to obtain 34.8g lithocholic acid. The yield is 87% and the purity is 95.1%.

[0047] Comparative Example 3

[0048] A method for refining lithocholic acid comprises the following steps:

[0049] 40g crude lithocholic acid (purity 90%±2%) was added to the reaction flask, and then 240mL of water was added, and a 10% sodium hydroxide solution was added dropwise at 10°C, and the pH was adjusted to 10 for reaction until the reaction solution was clear; after separation, 70mL of ethanol was added to the aqueous phase, and 1mol / L hydrochloric acid was added dropwise at 10°C to adjust the pH to 3, and when the precipitated solid no longer increased, the solid-liquid separation was performed, and the filter cake was washed with a small amount of methanol, and the solid was dried to obtain 34.6g of lithocholic acid. The yield was 86.5% and the purity was 95.6%.

[0050] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0051] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for refining lithocholic acid, characterized in that: The following steps are involved: The crude lithocholic acid reacts with a base in water, then dichloromethane is added to mix, an alcohol solvent is added to the aqueous phase after separation, and then an acid is added to adjust the pH to 3-4, solid is precipitated, and solid-liquid separation is performed to obtain lithocholic acid; Wherein, the base is at least one of potassium hydroxide, potassium carbonate, sodium hydroxide and sodium carbonate; The mass ratio of the crude lithocholic acid to the base is 1:1-2.5; The alcohol solvent is ethanol; The purity of the crude lithocholic acid is 85-95%; The crude lithocholic acid comprises a first compound having a structure of the following formula I and / or a second compound having a structure of the following formula II: (Formula I); (Formula II).

2. The method for refining lithocholic acid according to claim 1, characterized in that The acid is hydrochloric acid.

3. The method for refining lithocholic acid according to claim 1, characterized in that The temperature at which the crude lithocholic acid and the base react in water is 20-30°C.

4. The method for refining lithocholic acid according to any one of claims 1 to 3, characterized in that: The following steps are involved: The crude lithocholic acid is added to water, and the base is added at 20-30°C, and the pH is adjusted to 9-10 for reaction until the reaction solution is clear, and then dichloromethane is added for washing at least twice. After stratification, an alcohol solvent is added to the aqueous phase at a temperature of 10-20°C, and then an acid is added to adjust the pH to 3-4, a solid is precipitated, and solid-liquid separation is performed to obtain lithocholic acid.

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